This invention relates to methods for detecting nucleic acids.
Nucleic acid hybridisation is a widely used technique for identifying, detecting and quantitating target polynucleotide sequences in a sample. This technique relies for its success on complementary base pairing between the two halves of a double-stranded nucleic acid molecule: when single-stranded nucleic acids are incubated in solution under suitable conditions of temperature, pH and ionic strength, complementary base sequences pair to form double-stranded stable hybrid molecules. This ability of single-stranded nucleic acid molecules to form a hydrogen-bonded structure with their complementary nucleic acid sequences has long been employed as an analytical tool in recombinant DNA research.
In most cases the sample will contain double-stranded nucleic acid and must be denatured prior to the hybridisation assay to render it single-stranded. A nucleic acid having a known sequence which is complementary to the target sequence is either synthesised chemically in an automated fashion with great facility, or is isolated from the appropriate organism and rendered single-stranded by denaturation. It is then used as a probe to search a sample for a target complementary sequence. Detection of specific target nucleic acids enables accurate diagnosis of bacterial, fungal and viral disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence enables the diagnosis of human genetic disorders. Hybridisation produces stable hybrids, and a number of different approaches are known to the art for detecting these.
One approach involves the use of labelled probes. By labelling a probe nucleic acid with some readily detectable chemical group, it is possible to detect the polynucleotide sequence of interest in a test medium containing sample nucleic acids in single-stranded form. Nucleic acids have been labelled with radioisotopes, enzymes and fluorescent molecules. The use of labelled nucleic acids as probes in macromolecuiar analysis is important for clinical, veterinary and environmental diagnostic applications.
Early methods for detecting target nucleic acids involved their immobilisation on a solid support such as nitro-cellulose paper, cellulose paper, diazotized paper, or a nylon membrane. For example, in U.S. Pat. No. 4,358,535 to Falkow a method is disclosed in which the target nucleic acid is rendered single-stranded and then immobilised onto a membrane. A labelled probe which is complementary to the target nucleic acid is brought into contact with the solid support and hybridises to the target nucleic acid. The solid support is washed several times at a carefully controlled temperature to remove unbound and non-specifically bound probe without removing specifically bound probe, and the presence of the label in the resulting hybrid is determined. A disadvantage of this method is that it is neither easy nor convenient to attach the single-stranded target nucleic acid to a solid support, the whole process involving a 12-15 hour incubation of the nucleic acid with a nitro-cellulose sheet, followed by a 2 hour baking step. This makes the assay slow and unattractive for routine use. It is also cumbersome, with the hybridisation and washing steps being carried out in a sealed pouch, containing the membrane and the buffer solution. In addition, when very low concentrations must be detected, the ratio of specific to non-specificaily bound probe can be very low and repeated washing under highly stringent conditions is frequently required. Under these conditions the sensitivity of the assay is often compromised because of substantial loss of specifically bound probe.
Since then a many improvements have been made, most of which employ a sandwich approach using two probes: a reporter probe and a capture probe. The reporter probe is a nucleic acid having a sequence complementary to at least part of the target sequence and which is labelled with a detectable group. The capture probe is a nucleic acid having a sequence complementary to at least part of the target sequence, but which is different to that of the reporter probe, and which is labelled with an immobilisable group. In many applications, pairs of specific binding members (sbm""s) have been used for this purpose.
For example, in U.S. Pat. No. 5,273,882 to Snitman and Stroupe a capture probe complementary to part of the target nucleic acid is labelled with an antigen or antibody. After hybridisation between this capture probe and the target, the solution is introduced to a support-bound antibody or antigen which immobilises the hybrid formed between the capture probe and the target. Following a washing step, a second, reporter probe, complementary to a different region of the target nucleic acid, is introduced and the triple sandwich formed is detected.
Similar approaches are described by Holtke et al.: in U.S. Pat. No. 5,344,757 is disclosed a method in which a reporter probe is labelled with digoxin or digoxygenin, and hybrids are captured using antibodies against this hapten. In this case, a capture probe is not used, and the method is limited either to the detection of an immobilised target, or when the assay is used for detecting PCR products, one of the primers is immobilised. In U.S. Pat. No. 5,354,657 the method is further developed and involves the solution hybridisation between the target nucleic acid and a reporter probe labelled with digoxin or digoxygenin. This hybrid is captured by a solid-supported capture probe, complementary to a different region of the target. A detectably labelled antibody against the hapten is then added and the hybrids formed detected.
Specific binding members other than antigens or haptens and antibodies have been used. In U.S. Pat. No. 5,374,524 to Miller is described a method for the solution sandwich hybridisation, capture and detection of amplified nucleic acids. Amplicons are denatured and treated with an enzyme-labelled reporter probe and a biotinylated capture probe. Hybrids formed are captured using streptavidin-coated chromium dioxide particles.
Disadvantages of these approaches include the increased cost and complexity of using two probes. For example, for each assay two probes need to be synthesised and labelled: one for use as the capture probe, and the other for use as a reporter probe. In addition, hybridisation conditions have to be carefully chosen to form the sandwich of target, capture probe and reporter probe.
Simpler approaches which avoid the use of a capture probe have been described. Atlas and Steffan (Biotechniques (1990) 8:316-318) disclose a solution hybridisation method for detecting genetically-engineered micro-organisms in environmental samples. The detection method involves recovery of DNA from the microbial community of an environmental sample followed by hybridisation in solution with a radio-labelled RNA gene probe. After nuclease digestion of non-hybridised probe RNA, the DNA-RNA hybrids formed in the solution hybridisation are separated by column chromatography and detected by liquid scintillation counting. A less cumbersome approach is disclosed in U.S. Pat. No. 4,978,608 to Kung and Nagainis in which DNA is detected in a sequence non-specific manner using a high affinity single-stranded DNA-binding protein. This approach is extended in U.S. Pat. No. 5,536,648 to Kemp et al. who disclose an amplified DNA assay using a double stranded DNA binding protein. The method uses a PCR primer having a nucieotide sequence which is a ligand for a double stranded DNA-binding protein. After amplification the amplified target is captured by the double stranded DNA-binding protein immobilised on a solid surface. This method does not use a capture probe and will detect any amplification product containing the sequence which is a ligand for the double stranded DNA-binding protein. A disadvantage of this approach is that it relies an the accuracy of the amplification step for its specificity.
Another method is disclosed in U.S. Pat. No. 4,968,602 to Dattagupta. The test sample is modified chemically to introduce a reactive site. This mixture is then contacted with a reporter probe. After a solution phase hybridisation step, the hybrid is brought into contact with a surface having an immobilised reaction partner which reacts with the reactive site, and allows the unhybridised material to be washed away. A disadvantage of this approach is that the initial reaction step may interfere with the subsequent formation of the hybrid.
A further approach in which the hybrid itself is a hapten and which therefore only requires one probe is described by Carrico. In U.S. Pat. No. 4,743,535 is disclosed a nucleic acid hybridisation assay involving a reporter probe which results in the formation of a hybrid having epitopes for an antibody reagent. The antibody reagent is selective for DNA-RNA or RNA-RNA hybrids over the single-stranded nucleic acids. U.S. Pat. No. 5,200,313 to Carrico further discloses a nucleic acid hybridisation assay employing an immobilised or immobilisable polynucleotide probe selected to form DNA-RNA or RNA-DNA hybrids with the particular polynucleotide sequence to be determined. Resulting hybrids are detected by binding of an antibody reagent, preferably labelled with a detectable chemical group, selective for binding the hybrids in the presence of the single-stranded sample and probe nucleic acids.
Advantageous feature of Carrico""s inventions are that no immobilisation or labelling of sample nucleic acids is required and hybridisation can be performed entirely in solution. A further advantage is that a universal detection reagent may be used whatever the target is.
The key feature of Carrico""s invention is the requirement for antibodies specific for double-stranded hybrids having little affinity for single-stranded nucleic acid. The generation of specific polyclonal antibodies that will bind double-stranded nucleic acid but not single-stranded nucleic acid is complicated by the fact that polyclonal antisera may contain antibodies that will cross-react with single-stranded nucleic acid. Polyclonal antisera may also contain naturally occurring antibodies to single-stranded nucleic acid or antibodies to single-stranded nucleic acid arising as a result of the immunisation. Monoclonal antibody technology can provide a means to select an antibody with desired affinity and specificity which will overcome in part these problems. Such monoclonal antibodies which will selectively bind double-stranded DNA (U.S. Pat. No. 4,623,627) or DNA-RNA hybrids (U.S. Pat. No. 4,833,084 to Carrico) have been prepared. Monoclonal antibodies are however more expensive to produce and generally have lower affinities than polyclonal antibodies.
The monoclonal antibodies disclosed by Carrico (U.S. Pat. No. 4,833,084) are specific for DNA-RNA duplexes, particularly DNA-RNA heteropolymer duplexes, and are characterised by having cross-reactivity for binding to single or double-stranded DNA or RNA, as measured by competitive immunoassay, of less than about 1:1000, and preferably less than 1:10,000, and an affinity for DNA-RNA heteropolymer duplexes greater than 109 L/mol.
Chevrier et al. (Molecular and Cellular Probes (1993) 7: 137-197) report that up to 200 fmol of a capture probe may be attached to Covalink NH microwells (Nunc). The antibodies disclosed by Carrico would therefore have a lower detection limit of approximately {fraction (1/10,000)} of 200 fmol, or 20 amol. In addition non-specific binding between the labelled antibody and the surface on which the probe is immobilised will also contribute to a background signal. Carrico""s method is thus not applicable to the detection of very low concentrations of target nucleic acid which are in the range of sensitive detection systems such as signal amplification detection systems or chemiluminescence. The approach of Carrico finds utility in the detection of target amplification products, such as those generated by the polymerase chain reaction (PCR). For example, a commercial assay, GEN-ETI-K(trademark), from Sorin utilises probes immobilised on microtitre plates by means of a streptavidin-biotin bridge and antibodies against double stranded DNA labelled with peroxidase. Its chief application is in the assay of nucleic acid amplification products.
One approach to overcome the problem of cross-reactivity is disclosed in U.S. Pat. No. 5612,458 to Hyldig-Nielson and Pluzek They use antibodies to complexes between peptide nucleic acid (PNA) and nucleic acids, particularly antibodies to nucleic acid probe-DNA or nucleic acid probe-RNA hybrids.
Another approach is to attempt to improve the affinity or selectivity of the antibody used. Fliss et al. (Applied and Environmental Microbiology (1993) 59:2698-2705) disclose murine monoclonal antibodies specific for DNA-RNA hybrids which are used to detect Lysteria DNA-RNA hybrids formed in solution from a biotinylated gene probe and rRNA extracted from Lysteria. They also teach that the endonuclease digestion approach used by Atlas and Stefan (see above) does not efficiently separate hybridised from unhybridised molecules. Significantly, they do not teach that treatment with a nuclease to remove any single-stranded nucleic acids prior to capture with the murine monoclonal antibodies specific for DNA-RNA hybrids would offer an improvement to their assay.
In summary, Carrico discloses a simple method for capturing hybrids formed in solution between a target nucleic acid and a nucleic acid probe which utilises antibodies specific for double-stranded nucleic acid. A disadvantage of this approach is the cross-talk between antibody and any single-stranded nucleic acid which may be present. This limits the sensitivity of the assay. Atlas and Steffan disclose another solution phase hybridisation assay in which hybrids are separated by column chromatography following an endonuclease digestion step. A similar approach is utilised in nuclease protection assays. This is a sensitive technique for the detection, quantitation, and characterisation of RNA. The hybridisation reaction occurs in solution allowing complete hybridisation of the probe to the target mRNA.
After hybridisation, remaining single-stranded probe RNA and unhybridised sample RNA are removed by digestion with a mixture of ribonucieases A and T1, or S1 nuclease. Then, in a single step, the nucleases are inactivated and the remaining hybrids precipitated. Nuclease protection assays are not used for the detection of DNA. A disadvantage of these latter two methods is that they are cumbersome and do not accurately quantify the amount of target nucleic acid present.
There remains a need to combine the advantageous features of Carrico""s invention with one having reduced cross-talk, lower background and improved sensitivity.
In U.S. Pat. Nos. 4,683,195 and 4,683,202, DNA or RNA is amplified by the polymerase chain reaction (PCR). These patents are incorporated herein by reference in their entirety. This method involves the hybridisation of an oligonucleotide primer to the 5xe2x80x2 end of each complementary strand of the double-stranded target nucleic acid. The primers are extended from the 3xe2x80x2 end in a 5xe2x80x2xe2x86x923xe2x80x2 direction by a DNA polymerase which incorporates free nucleotides into a nucleic acid sequence complementary to each strand of the target nucleic acid. After dissociation of the extension products from the target nucleic acid strands, the extension products become target sequences for the next cycle. In order to obtain satisfactory amounts of the amplified DNA, repeated cycles must be carried out, between which cycles, the complementary DNA strands must be denatured under elevated temperatures.
A method of detecting a specific nucleic acid sequence present in low copy in a mixture of nucleic acids, called ligase chain reaction (LCR), has also been described. WO 89/09835 describes this method and is incorporated herein by reference in its entirety. Target nucleic acid in a sample is annealed to probes containing contiguous sequences. Upon hybridisation, the probes are ligated to form detectable fused probes complementary to the original target nucleic acid. The fused probes are disassociated from the nucleic acid and serve as a template for further hybridisation""s and fusions of the probes, thus amplifying geometrically the nucleic acid to be detected. The method does not use DNA polymerase.
Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh et al., Proc. Natl. Acad. Sci. (U.S.A.) (1989) 86:1173; Gingeras et al., WO 88/10315; Davey et al., EP 329,822; Miller et al., WO 89/06700), RACE (Frohman, In: PCR Protocols: A Guide to Methods and Applications, Academic Press, NY (1990)) and one-sided PCR (Ohara, et al., Proc. Natl. Acad. Sci. (U.S.A.) (1989) 86:5673-5677). Particularly suitable amplification procedures include Nucleic Acid Sequence-Based Amplification, Strand Displacement Amplification, and Cycling Probe Amplification.
Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting di-oligonucleotide, thereby amplifying the di-oligonucleotide, are also known (Wu et al., Genomics (1989) 4:560).
An isothermal amplification method has been described in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5xe2x80x2-[a-thio]triphosphates in one strand of a restriction site (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) (1992) 89:392-396).
It is important that enzymes employed as labels catalyse a reaction which has an easily detectable product, and have a high turnover number to allow sensitive detection: horseradish peroxidase and alkaline phosphatase are most common. Although sensitive chemiluminometric assays for horseradish peroxidase have been described which allow small amounts of enzyme to be detected, problems associated with its use include lack of enzyme and substrate stability and the presence of endogenous peroxidases in samples.
For alkaline phosphatase, enzyme amplification cycles have been described which further reduce the amount of enzyme which can be detected, thereby extending the detection limit. In one method, the amplification system comprises an apoenzyme which is convertible into a holoenzyme by interaction with an accessory subunit and a masked form of the subunit which is convertible into its active unmasked form by the action of the enzyme to be detected. For example, in U.S. Pat. No. 5,445,942 to Rabin et al., a method is disclosed for detecting a hydrolase enzyme able to hydrolyse a synthetic derivative of FAD substituted in such a way that it yields FAD when hydrolysed, and is incorporated herein by reference in its entirety. Here the subunit is FAD and the masked form is 3xe2x80x2FADP, and the apoenzyme is apoglucose oxidase or apo-D-aminoacid oxidase.
The FAD produced forms an active holoenzyme from the corresponding apoenzyme. This approach allows the detection of small amounts of alkaline phosphatase in short periods of time. For example, using such an amplification system in which the apoenzyme is apo-D-amino acid oxidase has permitted the detection of 0.1 amol of alkaline phosphatase in less than 30 minutes (Harbron et al., Anal. Biochem. (1992) 206: 119-124). In GB9622524.8 this approach is further extended to an amplification assay for nuclease P1, and is incorporated herein by reference in its entirety.
Broadly, the present invention combines advantageous aspects of the above techniques and discloses a new and improved method for detecting single-stranded target nucleic acid comprising the steps of:
(a) forming a hybrid between said target nucleic acid and a nucleic acid probe, said nucleic acid probe labelled with an enzyme reagent which hydrolyses single-stranded nucleic acid but is substantially without effect on double-stranded nucleic acid, said hybrid formed under conditions of pH which are outside the activity range of said enzyme reagent,
(b) adjusting said pH to a value within the activity range of said enzyme reagent,
(c) allowing said enzyme reagent substantially to hydrolyse any single-stranded nucleic acid present, and
(d) detecting said hybrid.
In a further aspect, the invention provides a variety of means for detecting the hybrid by means of a hybrid-binding reagent such as an antibody or DNA-binding protein specific for double-stranded nucleic acid, or by means of a pair or pairs of sbm""s. These may be a antigen or hapten and the corresponding antibody, biotin and avidin, streptavidin or neutravidin, or a nucleic acid binding protein specific for a sequence present in the nucleic acid probe. Any of these agents may be labelled with a detectable label which may an enzyme, a fluorescent moiety, a chemiluminescent moiety, an electro-chemiluminescent moiety or a coloured moiety.
In a further aspect the invention discloses a method for detecting DNA-RNA hybrids, DNA-DNA, RNA-RNA, DNA-RNA or DNA-PNA hybrids between a target nucleic acid and a nucleic acid probe having a sequence complementary to part of the target nucleic acid.
In a another further aspect the invention discloses a method for detecting hybrids between nucleic acid amplification products and a nucleic acid probe having a sequence complementary to part of the amplified nucleic acid.
In a further aspect the invention discloses a method for detecting hybrids between target nucleic acid extracted from a clinical specimen, a veterinary specimen, a food specimen or an environmental sample and a nucleic acid probe having a sequence complementary to part of the target nucleic acid.
In a another further aspect the invention discloses a method for the detection of multiple nucleic acid targets in a sample.
In further aspects the invention provides a kit for carrying out the method.
Preferred embodiments of the invention may enable one to achieve one or more of the following objects and advantages:
(a) to provide a method for detecting hybrids between a target nucleic acid and a nucleic acid probe having a sequence complementary to part of the target nucleic acid by means of a specific binding member, in which any single-stranded nucleic acid is removed by treatment with an enzyme reagent attached to said probe and which is specific for single-stranded nucleic acids. Advantages of the present invention are: only a single probe is required; highly sensitive detection systems, such as chemiluminescence or enzyme amplification cascades may be used to detect the hybrids; and the sensitive detection of target nucleic acid may be achieved without using target amplification techniques, such as PCR or LCR.
(b) to provide a method for detecting multiple nucleic acid targets. An advantage of the present invention is that a single sample may be screened for a number of targets, thereby increasing the speed of assay and reducing the number of sample which are required.
(c) to provide a universal method for detecting target nucleic acid. An advantage of the present invention is that it may be used with existing nucleic acid probes and their corresponding detection systems.
(d) to provide a method for detecting hybrids between DNA-RNA, RNA-DNA, RNA-RNA, RNA-PNA and DNA-PNA hybrids by appropriate selection of the hybrid binding reagent and enzyme reagent used.